Heart Vessels DOI 10.1007/s00380-014-0523-6

ORIGINAL ARTICLE

Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway Xiaowu Wang • Binbin Yuan • Wenpeng Dong Bo Yang • Yongchao Yang • Xi Lin • Gu Gong



Received: 29 September 2013 / Accepted: 9 May 2014 Ó Springer Japan 2014

Abstract Exposure to humid heat stress leads to the initiation of serious physiological dysfunction that may result in heat-related diseases, including heat stroke, heat cramp, heat exhaustion, and even death. Increasing evidences have shown that the humid heat stress-induced dysfunction of the cardiovascular system was accompanied with severe cardiomyocyte injury; however, the precise mechanism of heat stress-induced injury of cardiomyocyte remains unknown. In the present study, we hypothesized that humid heat stress promoted oxidative stress through the activation of angiotensin II (Ang II) in cardiomyocytes. To test our hypothesis, we established mouse models of humid heat stress. Using the animal models, we found that Ang II levels in serum were significantly up-regulated and that the Ang II receptor AT1 was increased in cardiomyocytes. The antioxidant ability in plasma and heart tissues which was detected by the ferric reducing/antioxidant power assay was also decreased with the increased ROS production under humid heat stress, as was the expression of antioxidant genes (SOD2, HO-1, GPx). Furthermore, we demonstrated that the Ang II receptor antagonist, valsartan, effectively relieved oxidative stress, blocked Ang II signaling pathway and suppressed cardiomyocyte apoptosis induced by humid heat stress. In addition, overexpression of antioxidant genes reversed cardiomyocyte apoptosis

X. Wang  B. Yuan  W. Dong  B. Yang  Y. Yang  X. Lin Center of Cardiovascular Surgery, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China G. Gong (&) Department of Anesthesiology, General Hospital of Chengdu Military Command, No. 270, Tianhui Road, Jinniu District, Chengdu 610083, Si Chuang, People’s Republic of China e-mail: [email protected]

induced by Ang II. Overall, these results implied that humid heat stress increased oxidative stress and caused apoptosis of cardiomyocytes through the Ang II signaling pathway. Thus, targeting the Ang II signaling pathway may provide a promising approach for the prevention and treatment of cardiovascular diseases caused by humid heat stress. Keywords Humid heat stress  Cardiomyocyte  Angiotensin II  Oxidative stress Abbreviations Ang II Angiotensin II AOA Antioxidant ability AT1 Angiotensin receptor 1 ELISA Enzyme linked immunosorbent assay FRAP The ferric reducing/antioxidant power assay GPx Glutathione peroxidase HHS Humid heat stress HO-1 Heme oxygenase 1 HRP Horseradish peroxidase JNK c-Jun amino-terminal kinases LDH Lactate dehydrogenase MAPKs Mitogen-activated protein kinases MDA Malondialdehyde NAD(P)H Nicotinamide-adenine dinucleotide phosphate qRT-PCR Quantitative real-time polymerase chain reaction RAS Renin–angiotensin system ROS Reactive oxygen species RT Room temperature SOD Superoxide dismutase TBS Tris-buffered saline TBST TBS and tween TPTZ Tripyridyl-s-triazine

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Introduction The heat load of a mammal’s body should be dissipated to maintain a constant body temperature, via a process called thermoregulation [1]. Activated peripheral and hypothalamic heat receptors that signal the hypothalamic thermoregulatory center and activate a series of responses can be induced by an increase in the temperature of the blood [2]. Heat stress causes serious physiological dysfunction that may result in heat-related diseases, including heat stroke, heat cramp, heat exhaustion, and even death [3]. Studies have shown that heat stress leads to an increase in metabolic demand and a reduction in splanchnic blood flow, which in turn induces intestinal and hepatocellular hypoxia; the hypoxia results in the generation of highly reactive oxygen and nitrogen species that accelerate mucosal injury [4, 5]. In addition, examination of muscle tissue, blood monocytes, and serum of persons subjected to heat stress confirms that such a heat stress response occurs in vivo [1, 6]. The cardiovascular system has been considered the primary target of heat stress [7, 8]. Generally, cardiac dysfunction and failure are the main causes of heatrelated death. For many years, the pathological mechanism of heat stress-induced cardiac dysfunction has been explored. Although it has been confirmed by several reports [9, 10] that the heat stress-induced dysfunction of the cardiovascular system was accompanied by severe cardiomyocyte injury, the pathway responsible for heat stress-induced injury of cardiomyocytes remains unknown. Involvement of the renin–angiotensin (Ang) system (RAS) in human pathophysiology has expanded to include several diseases beyond a traditional role in saltwater homeostasis [11]. RAS is a major determinant of arterial pressure and volume homeostasis in mammals through the action of the vasoactive peptide angiotensin II (Ang II) on vascular Ang II type 1 receptor (AT1R) [12, 13]. The activity of RAS, especially Ang II, increases in several cardiovascular diseases, such as myocardial infarction, myocarditis, cardiomyopathy, and hypertension [14, 15]. It has been established that many of the pathophysiological effects of Ang II are dependent on oxidative stress [16]. Numerous signaling pathways in response to Ang II are mediated by reactive oxygen species (ROS), mainly via NAD(P)H oxidase activation [17]. ROS, including superoxide and hydrogen peroxide, are able to regulate cysteinebased phosphatases, such as protein tyrosine phosphatases and lipid phosphatases, and directly influence cell signaling pathways [17]. Furthermore, Ang II is well known as an activator of mitogen-activated protein kinases (MAPKs), which are implicated in cell growth and hypertrophy, and MAPK activation, especially that of p38MAPK and c-Jun NH2-terminal kinase (JNK), is dependent on ROS [18, 19].

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The effect of humid heat stress on cardiomyocytes has not been elucidated yet. In the present study, we aim to investigate the promotion of oxidative stress and induction of apoptosis in cardiomyocytes by humid heat stress.

Materials and methods Adenoviral vectors The recombinant replication-deficient adenovirus Ad5E1 SOD-2/HO-1/GPx was generated by homologous recombination after cotransfecting 293 cells with PACCMVSOD-2/HO-1/GPx and a virus-rescuing vector pAdBHG10, as described [20]. The empty recombinant replicationdeficient adenovirus Ad5de170-3 was used as a control vector throughout the study. High titers of recombinant adenoviruses were amplified, purified, titered, and stored, as described [21]. Ventricular myocytes isolation and Ang II treatment Mouse ventricular myocytes were isolated according to a previous report [22]. Briefly, C57BL/6J mice of either sex were anesthetized with pentobarbital sodium (100 mg/kg ip) and the heart was removed. Within 3 min, the aortic opening was cannulated onto a Langendorff perfusion system [23] and heart was retrogradely perfused at a constant pressure of 55 mmHg for 5 min with a Ca2?-free bicarbonate-based buffer containing: 120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 5.6 mM glucose, 20 mM NaHCO3, 10 mM 2,3-butanedione monoxime, and 5 mM taurine, which was continuously gassed with 95 % O2 ? 5 % CO2. The enzymatic digestion was commenced by adding collagenase type II (Worthington, 0.5 mg/ml each) and protease type XIV (0.02 mg/ml) to the perfusion buffer and continued for 15 min. Ca2? (50 mM) was then added into the enzyme solution for perfusing the heart for another 10–15 min. The digested ventricular tissue was cut into chunks and gently aspirated with a transfer pipette for facilitating the cell dissociation. The cell pellet was resuspended for a 3-step Ca2? restoration procedure (i.e., 125, 250, 500 mM Ca2?). The isolated cardiomyocytes were then suspended in minimal essential medium (pH 7.35–7.45) containing 1.2 mM Ca2?, 12 mM NaHCO3, 2.5 % fetal bovine serum, and 1 % penicillin–streptomycin. Cultured cardiomyocytes were divided into five group: (1) control group; (2) cardiomyocytes were transfected with empty vector and treated with Ang II (100 ng/l); (3) cardiomyocytes were transfected with SOD-2 overexpression vector and treated with Ang II (100 ng/l); (4) cardiomyocytes were transfected with HO-1

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overexpression vector and treated with Ang II (100 ng/ l);(5) cardiomyocytes were transfected with GPx overexpression vector and treated with Ang II (100 ng/l). Animals Thirty 8-week-old C57BL/6J (B6) mice, weighing 20–22 g, were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). The animals were housed under standard conditions of 12-/12-h light/dark cycle at room temperature with routine diet and free access to water. All animal experimental procedures were conducted under the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in China. All efforts were made to minimize suffering. Experimental design To mimic humid heat stress, a hot chamber was used to create an environment with a designated temperature (40.0 ± 0.05 °C) and relative humidity (60 ± 5 %). A total of 30 mice were randomly divided into three groups: (1) ten mice administered with vehicle were held at room temperature (24.0 ± 1 °C) with relative humidity (45 ± 5 %); (2) ten mice administered with vehicle were held at high temperature (40.0 ± 0.05 °C) with relative humidity (60 ± 5 %) for 4 h per day; (3) ten mice administered with valsartan (Novartis Co., Ltd., Tokyo, Japan) were held at high temperature (40.0 ± 0.05 °C) with relative humidity (60 ± 5 %) for 4 h per day. In addition, valsartan was orally administered (10 mg/kg) using feeding needles according to previously reports [24]. Mice were administered with valsartan 2 h prior to humid heat stress. The blood pressures of mice were detected every week via a noninvasive tail-cuff system. The experiment continued for a total of 4 weeks. For experimental analysis, mice were sacrificed by the injection of sodium pentobarbital (Merck KGaA, Darmstadt, Germany 100 mg/kg). ROS evaluation To evaluate ROS production, three groups of mice were treated as above for 4 weeks and killed under deep anesthesia. The heart organs were quickly excised and frozen in liquid nitrogen. ROS were quantified in tissues by electron spin resonance spectroscopy with hydroxy-TEMPO.7. All measurements were performed in 2 parallel runs. ELISA analysis The levels of Ang II protein in serum were analyzed using a commercially available enzyme linked

immunosorbent assay (ELISA; Yanjin Biotechnology Co., Shanghai, China) according to the manufacturer’s procedure; the absorbance was taken at 450 nm using a 680XR Microplate reader (Biorad, Hercules, USA). All of the samples were analyzed in duplicate. The standard curve for Ang II estimation was created by linear regression analysis. FRAP: the ferric reducing/antioxidant power assay A working FRAP solution was prepared by mixing 10 volumes of 300 mmol/l acetate buffer (pH = 3.6) with one volume of 10 mmol/l TPTZ solution and one volume of 20 mmol/l FeCl3 solution. TPTZ was dissolved in 40 mmol/l hydrochloric acid. Serum samples were added in amounts of 10 ll and AOA was calculated as the absorbance increase from five parallel determinations. For the blank control, 20 ll of dH2O was added. Determination of oxidative stress The Biochemical Analysis Kit (Jiancheng Biotechnology Co., Nanjing, China) was used for the measurements of malondialdehyde (MDA) content, lactate dehydrogenase (LDH) release, and superoxide dismutase (SOD) activity according to protocol instructions. Western blot analysis Proteins from serum or tissue homogenate were collected and a total of 20–30 lg of protein was fractionated by 12 % SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK). The membrane was treated by shaking and blocking at room temperature (RT) with 2 % nonfat dry milk in Tris-buffered saline (TBS) for 1 h followed by incubation in primary antibodies (rabbit polyclonal AT1, SOD2, HO-1, GPx p38 MAPK, ERK1/2 and caspase-3 from Santa Cruz Biotechnology, Santa Cruz, CA, USA); samples were then diluted in blocking buffer (1:10,000) at 4 °C overnight and washed three times with TBS and Tween (TBST; 10 mM Tris–HCl, pH of 7.5, 150 mM NaCl, and 0.05 % Tween-20) for 10 min each time at room temperature. Subsequently, the membrane was incubated in peroxidase-conjugated secondary antibody goat anti-rabbit IgG (Boster Corporation, Wuhan, Hubei, China; diluted 1:3,000 in the blocking buffer) for 1 h. After washing three times with TBST and once with TBS each for 10 min, 1 ml of 4-chloro-1-naphthol (4CN) as a HRP substrate with 9 ml of TBS and 6 ll of H2O2 was used for visualizing the target protein in the dark for 5–30 min.

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Heart Vessels Table 1 List of primer sequences used

Gene

Sense primer

AT1

50 -GCACAATCGCCATAATTATCC-30

SOD2 HO-1

50 -CACCTATGTAAGATCGCTTC-30

0

0

50 -CGAGGGGCATCTAGTGGAGAA-30

0

0

50 -TTGAGCAGGAAGGCGGTCTTAG-30

5 -TTAGGGCTCAGGTTTGTCCAGAA-3

5 -ACTTTCAGAAGGGTCAGGTGTCC-3 0

GPx

5 -GGGCTCCCTGCGGGGCAAGGT-3

Caspase-3

50 -AATTCAAGGGACGGGTCATG-30

b-actin

0

0

5 -TCTGTGTGGATTGGTGGCTCTA-3

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted from cardiomyocytes using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Up to 5 lg of the total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Clontech, Palo Alto, CA, USA). The cDNAs were used as templates for qRT-PCR. b-actin was used as the control. The primer sequences are listed in Table 1. The qRT-PCR mixture system contained 5 ll SsoFastTM EvaGreen Supermix (BIO-RAD), 1 ll of cDNA (diluted in 1:50) and 2 ll of each of the forward and reverse primers (1 lM) to a final volume of 10 ll. The PCR procedure was as follows: 94 °C for 4 min; 94 °C for 20 s, 55 °C for 30 s, and 72 °C for 20 s; 2 s for plate reading for 35 cycles; and melting curve from 65 to 95 °C. b-actin was used as a control for normalizing gene expression. Experiments were performed independently at least three times. The data obtained were calculated by 2-DDCt and treated for statistical analysis as described previously [25], followed by analysis with an unpaired sample t test. Statistical analysis All experiments were performed independently at least three times. Differences between two groups were analyzed by Student’s t test. Differences between multiple groups were analyzed by ANOVA. A p value less than 0.01 was considered statistically significant.

Results Humid heat stress caused the up-regulation of Ang II and its receptor AT1 To investigate whether humid heat stress could act on the RAS, Ang II levels in the serum of the mice were measured by ELISA analysis. Results showed that the level of Ang II (42.1 ng/ml) was significantly up-regulated in the mice after humid heat stress exposure as compared to the normal control (19.2 ng/ml, p \ 0.05) (Fig. 1a). Furthermore, the

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Anti-sense primer

50 -ATGTACTTGGGGTCGGTCATG-30 50 -GCTTGTGCGCGTACAGTTTC-30 0

50 -CTGCTTGCTGATCCACATCTG-30

expression of AT1 receptor in cardiomyocytes was analyzed by qRT-PCR and Western blot. The results exhibited that AT1 mRNA (Fig. 1b) and protein expression levels (Fig. 1c, d) in cardiomyocytes were significantly up-regulated by humid heat stress. Humid heat stress decreased antioxidant ability in the serum and cardiomyocytes To explore the effect of humid heat stress on the antioxidant ability (AOA) in mice, the FRAP kit was used to study the AOA in the serum and cardiomyocytes. Results showed that the AOA both in the serum and in cardiomyocytes of the mice exposed to humid heat stress was markedly decreased compared with the control (Fig. 2). These results implied that humid stress might increase oxidative stress. Humid heat stress down-regulated the expressions of SOD2, HO-1 and GPx in cardiomyocytes Decreasing of AOA in cardiomyocytes may attribute to the increasing production of ROS. Reasonably, we evaluated ROS production in heart tissue of mice exposed to humid heat condition. Compared with the control, humid heat stress triggered obvious increase of ROS production (Fig. 3). To further confirm the effect of humid heat stress on oxidative stress, the expression of antioxidant genes including SOD2, HO-1 and GPx was analyzed in cardiomyocyte by qRT-PCR and Western blot. The qRT-PCR result showed that the mRNA expression levels of SOD2, HO-1 and GPx were dramatically down-regulated in the humid heat stress group compared with the control group (Fig. 4a). This result was further confirmed by Western blot (Fig. 4b, c). These results indicate that humid heat stress has a negative effect on antioxidant genes expression. Valsartan relieved oxidative stress induced by humid heat stress in cardiomyocytes To investigate whether the increased oxidative stress was regulated by Ang II, we studied the effect of the Ang II receptor antagonist, valsartan, on the levels of LDH, MDA

Heart Vessels Fig. 1 Analysis of the effect of humid heat stress on the expression of Ang II and its receptor AT1. a Analysis of Ang II in serum by ELISA; b qRT-PCR analysis of AT1 in cardiomyocytes; c, d Western blot analysis of protein levels of AT1 in cardiomyocytes; b-actin was used as a control. At least three independent experiments were performed. *p \ 0.01 vs. control denotes significant differences

Fig. 2 Determination of the variation of antioxidant ability in serum and cardiomyocytes. FRAP was performed according to the standard protocols; dH2O was determined as the blank control. At least three independent experiments were performed. *p \ 0.01 vs. control denotes significant differences

and SOD in cardiomyocytes. We found that humid heat stress significantly increased LDH release in cardiomyocytes. Pretreatment with valsartan, however, maintained these levels near baseline (Fig. 5a). Compared with the control group, SOD levels were also significantly downregulated under humid heat stress, which was markedly increased by pretreatment with valsartan (Fig. 5b). We also

Fig. 3 Electron spin resonance (ESR) analysis of hydroxy-TEMPO signal decay in the heart organs. *p \ 0.01 vs. control

observed that humid heat stress significantly increased MDA levels in cardiomyocytes compared with the control group, while pretreatment with valsartan significantly reduced MDA levels (Fig. 5c). The blood pressure of mice was also detected. Reduction of blood pressure was observed in mice pretreated with valsartan compared with humid heat stress group (Fig. 6). These results revealed that oxidative stress induced by humid heat stress could be relieved by pretreatment with the antagonist of the Ang II receptor.

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Fig. 4 Effect of humid heat stress on the expressions of SOD-2, HO1 and GPx. a Analysis of transcriptional levels of SOD-2, HO-1 and GPx in cardiomyocytes by qRT-PCR; actin was used as a control. More than three independent experiments were performed. b, c Western blot analysis of protein levels of SOD-2, HO-1 and GPx. b-actin was used as a control. More than three independent experiments were performed. *p \ 0.01 vs. control denotes significant differences

Valsartan blocked activation of Ang II pathway induced by humid heat stress MAP kinase activation has been proved to be involved in Ang II pathway. To confirm whether MAP kinase activation can be induced by humid heat stress, p-p38 MAPK and

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Fig. 5 Determination of the variation of LDH release, SOD and MDA in cardiomyocytes. a Variation of LDH release in cardiomyocytes; b variation of SOD in cardiomyocytes; c variation of MDA in cardiomyocytes. More than three independent experiments were performed. *p \ 0.01 vs. control denotes significant differences. # p \ 0.01 vs. HHS group denotes significant differences

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Fig. 6 Detection of blood pressure of mice model via a noninvasive tail-cuff system. ANOVA and Post hoc multiple comparisons were performed among these three groups in 7, 14, 21 and 28 days. Significant difference was found in 14, 21 and 28 days (p \ 0.01). In 14 days, HHS vs. control p \ 0.01, HHS vs. valsartan ? HHS p \ 0.01, valsartan ? HHS vs. control p [ 0.01; In 21 days, HHS vs. control p \ 0.01, HHS vs. valsartan ? HHS p \ 0.01, valsartan ? HHS vs. control p [ 0.01; In 28 days, HHS vs. control p \ 0.01, HHS vs. valsartan ? HHS p \ 0.01, valsartan ? HHS vs. control p [ 0.01

Fig. 8 Analysis of p-ERK 1/2 expressions of cardiomyocytes by Western blot. a Protein expression of p-ERK 1/2; b protein expression was analyzed using BandScan 5.0 software and normalized to b-actin, *p \ 0.01 vs. control, #p \ 0.01 vs. HHS

p-ERK1/2 expressions in cardiomyocytes of model mice were analyzed by immunoblot. Compared with the control, p-p38 MAPK and p-ERK1/2 expressions were significantly up-regulated by humid heat stress (Figs. 7, 8). However, pretreating with valsartan resulted in decreased expression. Valsartan inhibited cardiomyocyte apoptosis induced by humid heat stress To further confirm the reverse function of the Ang II receptor antagonist, the effect of valsartan on cardiomyocyte apoptosis was analyzed. We found that caspase-3, a key downstream effector protein of apoptosis, which is highly up-regulated by humid heat stress, was markedly down-regulated by valsartan (Fig. 9). Overexpression of antioxidant genes reversed cardiomyocyte apoptosis induced by Ang II Fig. 7 Analysis of p-p38 MAPK expressions of cardiomyocytes by Western blot. a Protein expression of p-p38 MAPK; b protein expression was analyzed using BandScan 5.0 software and normalized to b-actin, *p \ 0.01 vs. control, #p \ 0.01 vs. HHS

Humid heat stress caused increasing levels of Ang II in mice and caspase-3 expressions in cardiomyocyte. We next studied the role of antioxidant gene (SOD-2, HO-1, GPx) in the prevention of cardiomyocyte apoptosis induced by Ang

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Fig. 9 Analysis of transcriptional levels of caspase-3 in cardiomyocytes by qRT-PCR. Actin was used as a loading control. All experiments were independently performed at least three times. The data are expressed as the mean ± SEM and analyzed using the Student’s t test, and *p \ 0.01 is considered statistically significant

II. Mouse vascular cardiomyocyte was isolated and transfected with recombinant adenovirus. A dose (100 ng/l) of Ang II was added and co-cultured for 24 h. At the end of the treatment, cells were collected and cleavage caspase-3 expressions were analyzed by Western blot. The results showed that overexpression of antioxidant gene markedly reduced caspase-3 expressions in Ang II-treated cardiomyocyte (Fig. 10).

Discussion In general, we have demonstrated that humid heat stress may increase oxidative stress by Ang II and its receptor AT1 in cardiomyocytes. Humid oxidative stress inhibits antioxidant gene expression and induces the production of high levels of ROS which result in cardiomyocytes apoptosis. This study provides a mechanism of induction of ROS and Ang II in cardiomyocytes by humid heat stress. However, further investigation of the humid heat response in cardiomyocytes in vitro and in vivo is required. Humid heat stress causes extensive damage to organisms, including high blood pressure and multi-organizational damage [26, 27]. Researchers have confirmed that heat stress can cause apoptosis in myocardial cells and that the apoptosis rates were temperature-dependent. The RAS is a crucial part of the physiological and pathological responses of the cardiovascular system [16, 28, 29]. Ang II is the principal molecule of this system, and regulates vasoconstriction, the homeostasis of salt and water, cardiovascular hypertrophy and remodeling, and cardiomyocyte apoptosis [16]. Many studies have found that Ang II rapidly caused apoptosis in vivo in ventricular myocytes and non-myocyte

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Fig. 10 Analysis of expressions of cleavage caspase-3 in cultured cardiomyocytes by Western blot. a Protein expression of cleavage caspase-3; b protein expression was analyzed using BandScan 5.0 software and normalized to b-actin, *p \ 0.01 vs. control, #p \ 0.01 vs. mock

cells [30, 31]. In the present study, we observed that the levels of Ang II in the serum and its receptor AT1 in cardiomyocytes were remarkably increased after exposure to humid heat circumstances. Renal sympathetic nerve plays an important role in RAS activation [32]. These results suggest that humid heat stress may directly active renal sympathetic nerves [33, 34] which in turn modulate the activity of RAS and lead to an elevated level of Ang II. Furthermore, persistent Ang II stimulation may result in up-regulation of NFkappa B expression and increase transcription activity of AT1 receptor gene. It is now well established that ROS are potent inter- and intracellular second messengers [35, 36]. However, the excessive production of ROS may result in oxidative stress, loss of cell function, and ultimately apoptosis or necrosis. A balance between oxidant and antioxidant intracellular systems is, therefore, vital for cell function, regulation, and adaptation to diverse growth conditions [37]. Antioxidants within cells, cell membranes, and extracellular fluids can be up-regulated and mobilized to neutralize excessive and inappropriate ROS formation [38, 39]. Recent researches have shown that HHS could promote the levels of ROS in the body. One of the major sources of ROS is NAD(P)H oxidase located in smooth muscle cells [40]. Kimura et al.

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[41] have demonstrated that cardiac mitochondria are responsible for the acute Ang II-induced production of ROS, and NAD(P)H oxidase activation is essential because it leads to mitochondrial ROS production by Ang II stimulation. Living organisms are able to adapt to oxidative stress by inducing the synthesis of antioxidant enzymes and damage removal/repair enzymes [39]. Antioxidant enzymes (SOD2, HO-1 and GPx) play a vital role in protecting cellular damage from the harmful effects of ROS [42, 43]. In this study, humid heat stress exposure reduced the antioxidant ability in the serum and cardiomyocytes and down-regulated the expression of antioxidant genes (SOD2, HO-1 and GPx) in cardiomyocytes. Valsartan was highly effective in protecting cardiomyocytes from HHS-induced LDH release. Antioxidant enzyme activities (SOD) reflect the level of oxidative stress. Weak antioxidant capacity in the heart may be a factor that is responsible for the high sensitivity of this organ to humid heat stress-induced oxidative damage [44]. Increased MDA concentrations indicated that heat stress also caused oxidative stress, and increased red blood cell susceptibility to peroxidation. The biochemical determination of MDA indicates the formation of lipid peroxide [45]. Although the relationship between the events of cytotoxicity, ROS generation, and apoptosis induced by humid heat stress is not well defined, the ability of valsartan to reduce LDH release, MDA formation, and to up-regulate SOD activity may support the protective role of reducing cardiomyocyte apoptosis following humid heat stress. Apoptosis is the main form of cardiomyocyte death after heat stress, but the increase in oxygen-free radicals may lead to an important mechanism of cardiomyocyte apoptosis. Previous studies have shown that Ang II induces apoptosis in neonatal rat cardiomyocytes [46] and adult rat ventricular myocytes [24, 47]. Evidences have demonstrated that the AT1 receptor blocker inhibits cell apoptosis, implying that Ang II-induced myocyte apoptosis may be mediated via the AT1 receptor [48, 49]. In the present study, we confirm that Ang II induced by HHS causes apoptosis in cardiomyocytes, which is blocked by pretreatment with valsartan. In conclusion, our study demonstrated that humid heat stress up-regulates Ang II and its receptor AT1 which may play an important role in the induction of ROS and apoptosis in cardiomyocytes. However, the precise mechanism of humid heat stress on cardiomyocytes requires further investigation.

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Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway.

Exposure to humid heat stress leads to the initiation of serious physiological dysfunction that may result in heat-related diseases, including heat st...
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